Expression, purification, characterization, and solution nuclear magnetic resonance study of highly deuterated yeast cytochrome C peroxidase with enhanced solubility

Biochemistry. 2013 Apr 2;52(13):2165-75. doi: 10.1021/bi400220w. Epub 2013 Mar 21.

Abstract

Here we present the preparation, biophysical characterization, and nuclear magnetic resonance (NMR) spectroscopy study of yeast cytochrome c peroxidase (CcP) constructs with enhanced solubility. Using a high-yield Escherichia coli expression system, we routinely produced uniformly labeled [(2)H,(13)C,(15)N]CcP samples with high levels of deuterium incorporation (96-99%) and good yields (30-60 mg of pure protein from 1 L of bacterial culture). In addition to simplifying the purification procedure, introduction of a His tag at either protein terminus dramatically increases its solubility, allowing preparation of concentrated, stable CcP samples required for multidimensional NMR spectroscopy. Using a range of biophysical techniques and X-ray crystallography, we demonstrate that the engineered His tags neither perturb the structure of the enzyme nor alter the heme environment or its reactivity toward known ligands. The His-tagged CcP constructs remain catalytically active yet exhibit differences in the interaction with cytochrome c, the physiological binding partner, most likely because of steric occlusion of the high-affinity binding site by the C-terminal His tag. We show that protein perdeuteration greatly increases the quality of the double- and triple-resonance NMR spectra, allowing nearly complete backbone resonance assignments and subsequent study of the CcP by heteronuclear NMR spectroscopy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Circular Dichroism
  • Cloning, Molecular
  • Crystallography, X-Ray
  • Cytochrome-c Peroxidase / chemistry*
  • Cytochrome-c Peroxidase / genetics
  • Cytochrome-c Peroxidase / isolation & purification
  • Cytochrome-c Peroxidase / metabolism
  • Cytochromes c / metabolism
  • Escherichia coli / genetics
  • Gene Expression
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Nuclear Magnetic Resonance, Biomolecular
  • Protein Binding
  • Saccharomyces cerevisiae / chemistry
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Solubility
  • Spectrometry, Mass, Electrospray Ionization

Substances

  • Cytochromes c
  • Cytochrome-c Peroxidase